Developmental Morphology of Limb Reduction in Hemiergis (Squamata: Scincidae): Chondrogenesis, Osteogenesis, and Heterochrony

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1 JOURNAL OF MORPHOLOGY 254: (2002) Developmental Morphology of Limb Reduction in Hemiergis (Squamata: Scincidae): Chondrogenesis, Osteogenesis, and Heterochrony Michael D. Shapiro* Department of Organismic and Evolutionary Biology and Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts ABSTRACT Digit loss is acommon theme in tetrapod evolution that may involve changes in several developmental processes. The skink genus Hemiergis provides an ideal model to study these processes in closely related taxa: within three Western Australian Hemiergis species, digit quantity ranges between two and five. For three consecutive reproductive seasons, gravid females of Hemiergis were collected in the field and their embryos prepared for histological analysis of limb skeletal development (chondrogenesis and osteogenesis). Comparative studies of skeletal developmental morphology demonstrate that limbs with fewer than five digits do not result from a simple truncation of a putative ancestral (fivedigit) developmental program. The developmental and adult morphologies in two-, three-, and four-digit Hemiergis are neither predicted nor explained by asimple model of heterochrony involving either chondrogenesis or osteogenesis.inpostnatalhemiergis,digitnumberandrelative limb length do not correlate in asimple linear fashion. Instead, limb size and digit reduction may correlate with substrate conditions and burrowing behavior. J. Morphol. 254: , Wiley-Liss, Inc. KEY WORDS: Hemiergis; skink; limb development; limb reduction; digit; chondrogenesis; osteogenesis; heterochrony Digit loss is acommon theme in the evolution of many extant and extinct tetrapod groups (Sewertzoff, 1931; Gans, 1975; Alberch and Gale, 1985; Greer, 1991; Carroll, 1996). Among squamates, limb reduction definedasthephylogeneticlossofoneor morelimbskeletonelementsrelativetoanancestral morphology (Fig. 1) is especially frequent, occurring independently 62 times in 53 lineages (Greer, 1991). In skinks alone, reductions have occurred 31 times in 25 lineages (Greer, 1991). Lizards comprise ahighly diverse group of organisms, yet the patterns of digit loss within this group are remarkably conservative across taxa (and among tetrapods in general; Morse, 1872; Romer, 1966). In general, digits are lost in the following order:i V II III IV(Sewertzoff,1931;Greer, 1987, 1989, 1991). This conservatism may be rooted in convergent locomotor adaptations, shared developmentalconstraints,orperhapsboth.forexample, Greer (1991) proposes that evolutionary stability of the central digits may be correlated with aspects of autopod function: digits III and IV may be the most important in locomotion and thus selection favors their retention. Developmental factors may play amajor role in patterns of limb reduction as well. Amniote limb development programs appear to be highly conserved (Burke and Alberch, 1985; Shubin and Alberch, 1986; Shubin, 1991), which may severely constrain the potential range of adult morphologies. In general, limb skeletal condensations first appear proximally and yield other condensations through segmentation and bifurcation events. Most of these events occur along the posterior side of the limb, through the primary axis of condensation connectivity that includes the ulna (fibula in the hind limb), ulnare (calcaneum), distal carpal (tarsal) 4, and digit IV (Fig. 1; Burke and Alberch, 1985; Shubin and Alberch, 1986). This axis then curves anteriorlyandsequentiallyyieldsdigitsiii,ii,andi(the digital arch ). Burke and Alberch (1985) and Shubin and Alberch (1986) regard digit Vas aneomorphic structure that does not originate from the primary axis or digital arch. Alberch and Gale (1985) demonstrated that patterns of diversity in amphibianlimbsdependlargelyonthedevelopmentalproperties that characterize specific groups. Similarly, permutations of adult digit configurations in amniote limbs may be subject to developmental constraints imposed by the highly conserved primary axis and digital arch pattern of chondrogenesis Contract grant sponsors: The National Science Foundation; Contract grant number: Dissertation Improvement Grant IBN (toa.w.cromptonandmds);contractgrantsponsors:thesocietyfor Integrative and Comparative Biology, Sigma Xi, Robert A. Chapman Memorial Fellowship; The Putnam Expeditionary Research Fund of the MCZ, the Department of Organismic and Evolutionary Biology. *Correspondence to: Michael D. Shapiro, Department of Developmental Biology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA mshapiro@cmgm.stanford.edu. Published online 00 Month 2002 in Wiley InterScience ( DOI: /jmor WILEY-LISS, INC.

2 212 M.D. SHAPIRO (Holder, 1983; M.D. Shapiro and N.H. Shubin, unpubl. obs.). Despite a wealth of information on adult morphology in reduced-limbed squamates (Cope, 1892; Presch, 1975; Papenfuss, 1982; Choquenot and Greer, 1989; Greer, 1987, 1989, 1990, 1991; Tchernov et al., 2000, and references therein), the developmental origins of squamate limb reduction remain poorly understood. Developmental modes of digit loss in squamates may include evolutionary changes in ontogenetic rates or sequences. These changes can produce heterochronies, broadly defined as phyletic changes in morphology resulting from alterations of ancestral developmental timing (De Beer, 1930; Gould, 1977, 2000; Alberch et al., 1979). Several authors implicate truncation of an ancestral developmental program progenesis in the terminology of heterochrony (Alberch et al., 1979) as a mode of digit loss among squamates (e.g., Essex, 1927; Gans, 1975; Greer, 1987: partial truncations ), resulting in pedomorphic descendants (Müller, 1991). Other observations of adult reduced-limbed morphologies and general amniote limb development, however, predict that many limb configurations with fewer than five digits do not result from the simple truncation of ancestral pentadactyl ontogenetic trajectories (e.g., Shubin and Alberch, 1986; Greer, 1987, 1991). Nevertheless, neither hypothesis has been tested in a developmental framework. Fig. 1. Generalized manus and pes skeletons and primary axis of limb development in lizards. A: Distal forelimb skeleton of a lizard, beginning proximally with the distal ends of the ulna and radius, followed distally by the carpals and digits. Digits are numbered from anterior (digit I) to posterior (digit V). The primitive lizard phalangeal formula (number of phalanges per digit, beginning with the anterior digit) is for the forelimb (Romer, 1956). The primary axis of limb development (heavy shaded line) runs through the posterior elements of the limb and digit IV (Burke and Alberch, 1985; Shubin and Alberch, 1986). This axis subsequently curves anteriorly and, through a series of bifurcations and segmentations (narrow shaded lines), yields the anterior digits. B: Distal hind limb skeleton, beginning proximally with the distal tibia and fibula, followed by the tarsals and digits. The primitive lizard hind limb phalangeal formula is (Romer, 1956). The dashed oval in B indicates the approximate position of the embryonic calcaneum before fusion with the astragalus. Drawings are based on multiple specimens of the skink Glaphyromorphus gracilipes and are not to scale. Fig. 2. Adult manus and pes morphologies of (A) Hemiergis quadrilineata (2/2), (B) H. peronii (3/3), (C) H. peronii (4/4), and (D) H. initialis (5/5). For each morph, the manus is in the top row, pes in the bottom row. Anterior is to the left and distal is up in all panels. Phalangeal formulae are listed as described in Figure 1. Scale bars 1 mm. The Australian skink genus Hemiergis provides a rare chance to test these hypotheses in the context of closely related species. Limb reduction is common among Australian skinks and Hemiergis represents the best example of graded digit loss among humusdwellers (Choquenot and Greer, 1989; Greer, 1989, 1991). Within three closely related coastal Western Australian (WA) representatives, Hemiergis features a range of between two and five digits on the fore- and hind limbs. Hemiergis quadrilineata is a two-digit morph (two fingers / two toes, or 2/2), and H. initialis (5/5) has five digits (Fig. 2A,D). A third species, H. peronii, comprises two parapatric populations with fixed digit numbers: a three-digit western coastal population (H. peronii, 3/3) and a fourdigit population (H. peronii, 4/4) that inhabits the southern coast and inland forests (Choquenot and Greer, 1989) (Figs. 2B,C, 3). A principal aim of this study is to describe comparative patterns of limb skeletal chondrogenesis and osteogenesis in Hemiergis species and populations with different numbers of digits (hereafter referred to as different morphs ). This study of developmental morphology in Hemiergis will consider several questions about digit loss not approachable in distantly related traditional model species of tetrapod development (i.e., the mouse Mus musculus, the chicken Gallus domesticus, and the anuran Xenopus laevis). First, what differences in early skeletal ontogeny yield different numbers of digits in species of a single genus? To address this question, I present a detailed examination of chondrogenesis and osteogenesis in Hemiergis through serial sectioning and whole-mount clearing and staining of cartilage and bone. Second, based on these chondrogenesis and ossification data, does heterochrony

3 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS 213 Fig. 3. Map of localities sampled in this study. A: Map of southwest corner of Western Australia with collection areas for Hemiergis quadrilineata (2/2), Q; H. peronii (3/3), P3; H. peronii (4/4), P4; and H. initialis (5/5), I. B: Map of mainland Australia. The shaded rectangle represents the enlarged area in A. have any predictive or explanatory power in the context of limb reduction in Hemiergis? Heterochrony previously has been implicated in squamate digit loss (see above), but such hypotheses have never been tested directly. Third, what are the positional homologies of the digits in different limb configurations of Hemiergis? Adult digit homologies are easily discernible when five digits are present (as in H. initialis, 5/5), but not when one or more digits are missing (as in H. peronii, 3/3 and 4/4; and H. quadrilineata, 2/2). Studies of early digit development can help rectify this uncertainty. Fourth, in postnatal specimens, what is the relationship between digit number and relative limb length in Hemiergis? That is, do morphs with fewer digits also have shorter limbs relative to their body lengths? While instances of limb element loss and limb shortening have been documented extensively (see Greer, 1990; Gans, 1975, for reviews) the relationship between the two (i.e., whether limbs with fewer digits are also typically shortened) is poorly understood. Multiple regression analyses of limb and body lengths in Hemiergis will be used to test the hypothesis that digit loss is correlated with a decrease in overall limb size. MATERIALS AND METHODS Specimen Collection Hemiergis quadrilineata (2/2), H. peronii (3/3 and 4/4), and H. initialis (5/5) are live-bearing, semifossorial species that inhabit the semihumid to humid coastal shrublands and woodlands of southwestern WA (Fig. 3; an additional species not considered in this study inhabits a restricted, arid, inland region) (Choquenot and Greer, 1989; Greer, 1989; Storr et al., 1999; Cogger, 2000). Gravid Hemiergis females were wild-caught between 1997 and 2000; collecting expeditions began in October or November of each year and ended in January of the next. All animals were collected by hand in leaf litter or under logs, rocks, sheet metal, and other debris. Captured animals were transported to the Western Australian Museum (WAM; Perth, Australia) and were either sacrificed within 24 h for embryo processing or were held in captivity in glass aquaria lined with 2 8 cm of moist sand and leaf debris. Captive gravid lizards that were not sacrificed by the end of each expedition were transported alive to the Museum of Comparative Zoology (MCZ; Cambridge, MA) to allow further embryo development and harvesting. While in captivity, lizards subsisted on a diet of termites and small mealworms at the WAM or pinhead crickets and mealworms at the MCZ. Embryo Harvesting and Fixation For embryo harvesting, gravid females were sacrificed and a longitudinal incision was made along the ventral abdomen, beginning just posterior to the sternum and extending to the anterior tips of the pubes. Oviducts containing eggs were removed and submerged in either phosphate-buffered saline (PBS) or 0.4% aqueous sodium chloride for dissection. Embryos were removed from their eggs, staged (Dufaure and Hubert, 1961; translated by Porter, 1972), fixed overnight at 4 C in either Dent fixative (20% dimethyl sulfoxide in methanol; Dent et al., 1989) or 4% paraformaldehyde in PBS, then dehydrated through a graded methanol series and stored in 100% methanol at 20 C. Some late (stage 36 and higher) embryos were fixed in 10% neutral-buffered formalin (NBF) and stored in 70% ethanol to optimize bone staining. Adult specimens were fixed in 10% NBF and stored in 70% ethanol.

4 214 M.D. SHAPIRO TABLE 1. Forelimb chondrogenesis and ossification matrices for Hemiergis quadrilineata (2/2), H. peronii (3/3 and 4/4), and H. initialis (5/5) Phalanges Distal carpals Metacarpals I II III IV V I II III IV V II III IV V III IV IV Stage Specimen SVL H R U re ue c Fusions H. quadrilineata (2/2) / / J HQ dc4-dc5, ue-c J HQ dc4-dc5 J HQ dc4-dc5 J HQ dc4-dc5 J HQ dc4-dc5 J HQ dc4-dc5 J HQ dc4-dc5 H. peronii (3/3) A A ? 3? 2? / / / A / B J HT J HT J HT S HT H. peronii(4/4) / / ? J J J HP J HP J HP J HP H. initialis (5/5) / NA 3? 3? 3?

5 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS 215 TABLE 1. (Continued.) Phalanges Distal carpals Metacarpals I II III IV V I II III IV V II III IV V III IV IV Stage Specimen SVL H R U re ue c Fusions 35/ NA ? 3? 3? ? 3? 3? dc4-dc5 J HI J HI J HI dc4-dc5 J HI dc4-dc5 J HI dc4-dc5 J HI dc4-dc5 J HI dc4-dc5 J HI dc4-dc5 J HI J HI dc4-dc5 S HI dc4-dc5 S HI dc4-dc5 A HI dc4-dc5 A HI dc4-dc5 For each element, 0 absent, 1 prechondrogenic, 2 chondrogenic, 3 ossified, and 4 secondary centers of ossification present (long bones only); question marks (?) after a number indicate ambiguous staining; elements not normally present in a given morph are indicated with dashes ( ); snout vent length (SVL) is rounded to the nearest millimeter for most embryonic specimens; all 5-digit specimen numbers are MCZ A series. J, juvenile; S, subadult; A, adult; H, humerus; R, radius; U, ulna; re, radiale; ue, ulnare; c, centrale; dc, distal carpal; phalanges are listed by digit number and proximodistal position (e.g., II-3 is the third phalanx of the second digit). Clearing and Staining of Cartilage and Bone Developmental series from each morph were cleared and stained as whole mounts using a modification of a standard technique to visualize chondrogenesis and ossification patterns (Dingerkus and Uhler, 1977). Modifications included a lower trypsin concentration ( %) for overnight treatment for small embryos (stage 34 and earlier) and embryo bleaching (when necessary) in 1 l/ml hydrogen peroxide in 25% glycerol (in 0.5% aqueous potassium hydroxide). A dark alizarin red stain was achieved using a % solution in 2% potassium hydroxide. Skeletal condensations that stained with Alcian blue were scored as present and prechondrogenic tissues were scored separately; structures composed of the latter were very faint and lacked the typical chondrocyte morphology visible in whole mount at high magnification (though it is possible that some of these elements were indeed very early chondrogenic). Whole-mount observations were occasionally verified through stained serial sections (Hall, 1986; Celestine blue step omitted). Limb skeleton elements stained with alizarin red were scored as ossified and secondary (epiphyseal) centers of long bone ossification were scored if red stain localized within the cartilaginous epiphyses. Advanced embryos often did not stain successfully with alizarin red; most of these specimens were either fixed in Dent fixative or stored for an extended time in methanol. In these instances, unstained bones were scored as ossified if bone was clearly visible through microscopy. Clearing and staining of postnatal specimens was performed as described (Dingerkus and Uhler, 1977) but with the alizarin red staining modifications noted above. Regression Analysis of Limb Lengths Differences in postnatal limb lengths between the four Hemiergis morphs were assessed by multiple regression analysis controlling for snout-vent length (SVL). Specimens from the collecting expedition were measured for SVL and limb length defined here as the measurement from the body wall to the tip of digit IV normal to the body axis at the time of sacrifice (i.e., before fixation). Left limbs were measured unless they were missing or incomplete. Only female adults were measured, but neonates and juveniles of both sexes were measured. A total of 20 H. quadrilineata (2/2; SVL range mm), 66 H. peronii (3/3; mm), 77 H. peronii (4/4; mm), and 51 H. initialis (5/5; mm) were measured. Fore- or hind limb length was used as the dependent variable for each analysis and SVL and three dummy variables (H. initialis, 5/5, and the two H. peronii morphs) were used as independent variables. Hemiergis quadrilineata (2/2) was used as the default variable. RESULTS Condensation of the Limb Skeletons Stylopod and zeugopod. Limb element condensations and ossifications are summarized in Tables 1 and 2. Early stages of Hemiergis limb development closely resemble known ontogenetic sequences of pentadactyl lizards (Sewertzoff, 1904, 1931; Mathur and Goel, 1976) and amniotes in general (Holmgren, 1933; Burke and Alberch, 1985; Shubin and Alberch, 1986) (Fig. 4). Condensations appear from proximal to distal, beginning with the stylopod elements (forelimb: humerus; hind limb: femur), which are present as cartilage by stage 31. The zeugopod elements (forelimb: radius and ulna; hind limb: tibia and fibula) condense and chondrify by the end of stage 32. In contrast with the branching Y pattern of chon-

6 216 M.D. SHAPIRO TABLE 2. Hind limb chondrogenesis and ossification matrices for Hemiergis quadrilineata (2/2), H. peronii (3/3 and 4/4), and H. initialis (5/5) Phalanges Stage Specimen SVL Fe T Fi as ca dt s Metatarsals I II III IV V I II III IV V II III IV V III IV IV Fusions H. quadrilineata / as-ca 36/ as-ca as-ca J HQ as-ca J HQ as-ca J HQ as-ca J HQ as-ca J HQ as-ca J HQ as-ca J HQ as-ca H. peronii (3/3) A as-ca A as-ca as-ca ? 3? 3? as-ca 36/ as-ca 38/ as-ca 38/ A as-ca as-ca 38/ B as-ca J HT as-ca J HT as-ca J HT as-ca S HT as-ca H. peronii (4/4) as-ca as-ca ? 2 2 1? 0 1? as-ca as-ca as-ca as-ca 37/ as-ca as-ca as-ca 38/ as-ca as-ca as-ca as-ca J as-ca J as-ca J HP as-ca J HP as-ca J HP as-ca J HP as-ca H. initialis (5/5) / NA as-ca

7 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS 217 TABLE 2. (Continued.) Stage Specimen SVL Fe T Fi as ca dt s Metatarsals Phalanges I II III IV V I II III IV V II III IV V III IV IV Fusions 35/ NA as-ca as-ca as-ca as-ca as-ca as-ca as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca J HI as-ca S HI as-ca S HI as-ca S HI as-ca Fe, femur; T, tibia; Fi, fibula; as, astragalus; ca, calcaneum; dt, distal tarsal; numbering system and phalangeal abbreviations as for Table 1. drogenesis observed in some tetrapods (Burke and Alberch, 1985; Shubin and Alberch, 1986), the zeugopod elements of Hemiergis condense separately. Carpals. Mesopodial development along the amniote primary axis is characterized by bifurcation and segmentation events, whereas anterior elements (i.e., from the radius) exhibit segmentation only (Shubin and Alberch, 1986; Müller, 1991). In the forelimb of Hemiergis, the ulnare (ue), centrale (c), and distal carpals (dc) 1 4 originate from the primary axis; the radiale (re) and dc5 likely do not. In several specimens, two faint condensations were visible in the re position early in development (also noted by Müller, 1991). Table 3 outlines the orders of carpal appearance in the different Hemiergis morphs. Other skeletal elements of the hand include the pisiform (pis), which is likely a sesamoid bone that does not originate from a segmentation or bifurcation event (Shubin and Alberch, 1986; Rieppel, 1992a). An additional small cartilage appears between the distal radius and ulna at stage 38 or later in some specimens and is ossified in some adults (e.g., Fig. 2A,C). The position occupied by this small bone suggests that it is the intermedium, but its late appearance suggests that it may be a sesamoid element. The latter scenario would be supported if the element lies within a tendon, but this possibility has not been pursued. TABLE 3. Carpal condensation sequences in Hemiergis Fig. 4. Shared limb skeleton condensation configurations in Hemiergis. In this schematic diagram, elements present in all morphs at a given stage appear as blackened shapes, elements exhibiting variability appear in gray. Younger stages are to the left of the figure, older ones are to the right. A: Forelimb condensations first appear proximally and include the humerus, radius, and ulna. Digits II V appear in the order, IV III V II. Phalangeal condensation begins with digit IV. B: Hind limb elements appear in a similar sequence, beginning with the femur, tibia, and fibula. Fe, femur; Fi, fibula; H, humerus; R, radius; T, tibia; U, ulna; digits I V are indicated by roman numerals. Species Order of condensation appearance H. quadrilineata (2/2) ue dc4 dc3 (c, re) (dc5, dc2) pis H. peronii (3/3) ue dc4 dc3 dc5 c re dc2 pis H. peronii (4/4) ue dc4 dc3 c re dc5 dc2 pis H. initialis (5/5) ue dc4 c (re, dc3) dc2 (dc1, dc5) pis

8 218 M.D. SHAPIRO Fig. 5. Coronal section of a stage 33 Hemiergis quadrilineata (2/2) (MCZ A10422) hind limb showing incipient fusion of the astragalus and calcaneum. A: Fusion begins with mesenchymal connectivity (arrowhead), followed by chondrogenesis at later stages (magnification 20 ). No mesenchymal connectivity between the as and ca is observed until this stage. B: Magnification of boxed area in A, 50. as, astragalus; ca, calcaneum; dt, distal tarsal; Fi, fibula; mt, metatarsal; T, tibia; digits II V indicated by roman numerals in A. Fig. 6. Skeletal condensations along the primary axis in Hemiergis at stages 32 and 33. A,B: Stage 32 forelimb (A) and hind limb (B) of H. peronii (4/4) (MCZ A38061) cleared and stained with Alcian blue. Cartilaginous elements appear in the stylopodia and zeugopodia of both limbs and faint autopodial condensations representing presumptive digit IV are also visible in both limbs. Serial sections of similarly aged limbs reveal only mesenchymal condensations in the autopod (not shown), and hence the digits in A and B are likely not cartilaginous. C: Coronal section of a stage 33 H. peronii (4/4) (MCZ A38073) hind limb. The primary axis of limb skeletal condensation passes through the fibula, calcaneum, distal tarsal 4, and metatarsal 4. The first phalangeal condensation to appear in the fore- and hind limbs of all Hemiergis morphs studied is the proximal phalanx of digit IV (arrowhead). Only mesenchymal portions of metatarsals 2 and 5 are visible in this section. as, astragalus; ca, calcaneum; dt, distal tarsal; Fe, femur; Fi, fibula; H, humerus; IV, digit IV condensation; mt, metatarsal; R, radius; T, tibia; U, ulna.

9 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS Fig. 7. Stage 33 embryos of Hemiergis. Embryos of (A) H. quadrilineata (2/2), (B) H. peronii (3/3), (C) H. peronii (4/4), and (D) H. initialis (5/5) have similar external morphologies at this and earlier stages. e, eye; fl, forelimb; hl, hind limb; mb, midbrain; t, tail. Scale bar 1 mm. Tarsals. Tarsal condensation sequences are more similar across species, perhaps owing to the presence of fewer elements. The reptilian calcaneum (ca) and distal tarsals (dt) 3 and 4 originate from the primary axis. The anterior proximal tarsal, however, may originate from the intermedium and other condensations (for a discussion of homologies, see Holmgren, 1933; Schaeffer, 1941; Mathur and Goel, 1976; Burke and Alberch, 1985; Shubin and Alberch, 1986). In Hemiergis, only one condensation was discernible directly anterior to the ca, but the embryonic connectivity of this element is ambiguous. Following traditional nomenclature, this element is here referred to as the astragalus (as). The order of tarsal appearance in all Hemiergis morphs examined is ca dt4 as dt3. Carpal and tarsal fusions. Cartilaginous fusions were frequently observed in the fore- and hind limbs of Hemiergis. In the forelimb, most H. quadrilineata (2/2) and H. initialis (5/5) specimens exhibited a fused dc4 and dc5 ( dc4 5 ) at, or just before, parturition (Table 1). Fusion (or perhaps nonsegmentation) of the ue and c was also observed in a single juvenile specimen (HQ-004) of H. quadrilineata (2/2). In the hind limb, cartilaginous fusion of the as and ca begins in all morphs between stages 33 and 34 (Fig. 5), yielding a single tarsale proximale (Sewertzoff, 1931: 135) by stage Digit condensations: shared developmental configurations. Metacarpal and metatarsal 4 the first digit elements to appear in the fore- and hind limb, respectively invariably condense along the 219 primary axis in amniotes (Burke and Alberch, 1985; Shubin and Alberch, 1986; Müller and Alberch, 1990). The axis of digital development then curves anteriorly and sequentially yields digits III and II. Digit V appears temporally after digit III but before digit II in Hemiergis (Fig. 4). Phalanges are added to each digit in Hemiergis in the order of appearance of their supporting metacarpals or metatarsals. Hence, the first phalanx to appear in each limb is the proximal phalanx of digit IV (IV-1) (Fig. 6), yielding a phalangeal formula of X (where X denotes absence of a digit and 0 indicates a metapodial but no phalanges). The addition of phalanx IV-1 to both sets of limbs during stage 33 marks the final common configuration in the development of the four morphs. Hemiergis initialis (5/5) was not represented by specimens with this specific morphology but, based on digit configurations (and relative Alcian blue staining intensities) of stage 33 and 34 embryos, this species likely also passes through a configuration with four metacarpals/metatarsals and one phalanx on digit IV. At this final common phase of limb skeletal morphology, Hemiergis embryos are similar externally as well: all show similar degrees of development of the lower jaw, eyes, endolymphatic sacs, and (closed) branchial slits (Fig. 7). The limbs, too, are similar in external view and appear to contain the early condensations of four digits in both fore- and hind limbs; this similarity is especially pronounced between the two H. peronii (3/3 and 4/4) morphs (Fig. 8). Phalangeal condensations: five-digit sequence. Following the above series of shared configurations, the limb skeletal developmental sequence of Hemiergis initialis (5/5) diverges from those of H. peronii (3/3 and 4/4) and H. quadrilineata (2/2) during stage 34. In addition to a divergence in skeletal developmental sequences, the overall shapes of the limb paddles differ between the four morphs as well (Fig. 9). At stage 34, H. initialis (5/5) adds mc1 and mt1, elements completely lacking in the other taxa (Fig. 10). Phalangeal addition also continues across all digits not one digit at a time until the adult configuration ( ) is reached in both sets of limbs. Although H. initialis has five digits, its phalangeal configuration is slightly reduced from the primitive squamate morphology: on both the hands and feet, digit IV has only four phalanges, compared to the five present in the primitive squamate configuration (Figs. 1, 2D). Likewise, pedal digit V is missing a phalanx for an adult total of three. Four-, three-, and two-digit sequences. The following results focus upon the forelimbs of Hemiergis quadrilineata (2/2) and H. peronii (3/3 and 4/4) (Fig. 11A), but hind limb sequences are nearly identical (Fig. 11B). After a shared digit configuration with single phalanges on digits III and IV, the two-, three-, and four-digit developmental sequences di-

10 220 M.D. SHAPIRO Fig. 8. Morphological divergence of three- and four-digit morphs of Hemiergis peronii. In all panels, digits II V are indicated by roman numerals; anterior is to the left and distal is up. All limbs are shown in dorsal view. A D: Stage 33 limbs of (A,C) H. peronii (3/3) and (B,D) H. peronii (4/4). At this stage, four digital condensations are visible externally in both the forelimbs (A,B) and hind limbs (C,D) of each morph. E,F: Forelimbs cleared and stained for cartilage verify four metacarpal condensations at this stage, with a single phalanx (arrowhead) over digit IV in each limb. G,H: Cleared and stained hind limbs reveal a similar condensation patterns. I L: At stage 34, the proximal phalanx of digit V (arrowheads) appears in the four-digit morph (J,L), but not in the three-digit morph (I,K). verge (Fig. 11; also see Fig. 8I L). Hemiergis peronii (4/4) adds phalanges to the second, fourth, and fifth digits to bring the phalangeal formula to X The proximal phalanges of digits II and V are added last to achieve this configuration, with IV-2 added slightly earlier. Phalanges subsequently condense proximally to distally across all four metacarpals and metatarsals until the adult phalangeal configuration of X is attained. After the shared X configuration, digit V is excluded from the skeletal developmental program of Hemiergis peronii (3/3), and digits II and V from H. quadrilineata (2/2). Hemiergis peronii (3/3) adds phalanges across digits II, III, IV until the adult formula of X is reached. Hemiergis quadrilineata (2/2) not only has a reduced number of digits, but also fewer phalanges than expected for digits III and IV: relative to the primitive squamate condition, single phalanges are lost from the two remaining digits for an adult formula of X Ossification of the Limb Skeletons Stylopod and zeugopod. Whole-mount staining detects ossification of the stylopod element between stages in all morphs, followed shortly thereafter by zeugopod ossification (Tables 1, 2). The relative intensity and extent of staining indicate that the anterior zeugopod elements (forelimb: radius, hind limb: tibia) ossify before the posterior ones. However, serial sections of stage 33 and 34 embryonic limbs suggest that ossification begins earlier than is detectable by whole-mount alizarin staining. In Hemiergis peronii (4/4), for example, early perichondral ossification is observed in the humerus of a stage 33 forelimb (MCZ A38073) and the femur, tibia, and fibula of an H. peronii (3/3) stage 34 limb (MCZ A38117). Secondary centers of ossification form in the long bone epiphyses postnatally, beginning with the stylopod elements (Table 2). In specimens that have secondary centers in only one zeugopod element (HQ-003, HQ-005, HP-005, HP-006, HI-006), the posterior element always dominates. Carpals and tarsals. Carpal and tarsal ossification in Hemiergis also begins postnatally and carpal ossification lags behind the tarsus (also noted in other lizards by Rieppel, 1992b, 1993a, 1994a). In general, carpal ossification begins on the posterior

11 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS Fig. 9. Stage 34 Hemiergis limb paddles and skeletal condensations. A D: Forelimbs are in the top row, hind limbs in the bottom row; anterior is to the left and distal is up. By this stage, the different morphs have divergent paddle shapes and condensation patterns. A: Limbs of H. quadrilineata (2/2) based on MCZ A B: Limbs of H. peronii (3/3) based on MCZ A C: Limbs of H. peronii (4/4) based on MCZ A D: Limbs of H. initialis (5/5) based on MCZ A In A and B, metacarpals and metatarsals that will not support phalanges have less mesenchyme distal to them (arrowheads). Dashed outlines indicate precartilaginous elements. Scale bar (D) 0.5 mm. 221 secondary ossification centers in lizard tarsals, see Dollo, 1884, and Mathur and Goel, 1976.) Digits. Digit ossification begins late in embryogenesis in all four morphs (Tables 1, 2) and shaft ossification was complete or well underway in the metapodials and phalanges of all neonates examined. Early signs of cartilage hypertrophy appeared first in the hind limb, beginning with the metatarsals (mt) in the order mt4 mt3 mt2 ( mt1 in Hemiergis initialis, 5/5). Metatarsal 5 cartilages hypertrophied and ossified last in all morphs. Phalangeal hypertrophy begins with IV-1, III-1, and IV-2, which also showed the most advanced ossification across taxa. In general, ossification of the remaining phalanges occurred from proximal to distal, although ungual phalanges stained more intensely with alizarin red than did penultimate ones in some late-stage embryos. The forelimbs of all morphs follow a similar ossification sequence, although bone formation lags slightly behind the hind limb. Secondary centers of ossification appear in the metacarpals and metatarsals postnatally. side of the limb with the ue and dc4, followed by the re (Table 4). Carpal ossification is variable, however, in juvenile H. initialis (5/5; n 10). In specimens with ossified carpals (n 3), two (HI-006, HI-101) showed partial ossification of the re with no ossification or cartilage hypertropy in other carpals. A third specimen (HI-002) showed dominance of the posterior carpals with an ossified ue and hypertrophied dc4 portion of dc4 5. A fourth (HI-105) showed no carpal ossification but hypertrophy of the ue and dc4. In the forelimbs of five Hemiergis quadrilineata (2/2) neonates, the portion of dc4 5 proximal to mc4 stained with alizarin red while the portion supporting mc5 did not (Fig. 12A). A larger specimen (HQ- 101), however, revealed separate ossification centers in each of these distal carpals (Fig. 12B). Hence, these elements ossified separately, despite having developed from a single cartilage (also observed in the salamander Thorius; J. Hanken, pers. commun.). Tarsal ossification sequences are consistent between different Hemiergis morphs and occur in the following order: as ca dt4 dt3. Although the as and ca fuse as cartilages, these two elements ossify separately (Sewertzoff, 1908; Mathur and Goel, 1976; Rieppel, 1992a, b). Ossification of the as begins in a similar position to its initial site of chondrogenesis, just distal to the medial edge of the tibia (Fig. 12C). In several specimens, secondary centers of ossification formed in the as, ca, and dt4 as the primary ossifications approached the edges of their respective cartilages (Fig. 12D). (For a discussion of Fig. 10. Digit condensation sequence in Hemiergis initialis (5/5). Far left of A,B: Schematic diagrams of shared (A) fore- and (B) hind limb morphologies in all morphs of Hemiergis. Cartilaginous elements appear in black, precartilaginous in gray. Following this shared digit configuration, H. initialis (5/5) diverges from H. peronii (3/3 and 4/4) and H. quadrilineata (2/2). A: Forelimb condensation sequence. In H. initialis (5/5), metacarpal 1 is added last, along with phalanges over digits III, II, and V. Phalanges are subsequently added across all five digits until the adult phalangeal formula of is attained. B: Hind limb condensation sequence. The pattern of phalangeal addition is similar to that of the forelimb. The adult hind limb phalangeal formula is identical to that of the forelimb.

12 222 M.D. SHAPIRO Regression Analysis of Limb Lengths Multiple regression analysis of limb lengths reveals significant differences among morphs (P for fore- and hind limb analyses) and these differences do not correlate in a simple linear fashion with digit numbers (Fig. 13). Relative to body length, Hemiergis peronii (4/4) has the longest foreand hind limbs, followed by H. peronii (3/3). Hemiergis initialis (5/5) and H. quadrilineata (2/2) have relatively shorter limbs than both H. peronii (3/3 and 4/4) morphs, but their positions in the length Species TABLE 4. Carpal ossification sequences in Hemiergis Order of ossification H. quadrilineata (2/2) ue dc4 re (dc3, pis) c dc5 dc2 H. peronii (3/3) ue dc4 (re, c, dc5, dc3, pis) dc2 H. peronii (4/4) ue dc4 re (c, dc3) (dc5, dc2, pis) H. initialis (5/5) ue dc4 re (c, dc5, dc3, dc2, pis) dc1 gradient vary between the forelimb and hind limb. In terms of digit number, the gradient of forelimb length (from longest to shortest) is (Table 5). In the hind limb, the gradient is (Table 6). DISCUSSION Fig. 11. Comparison of digit condensation sequences among two-, three-, and four-digit morphs of Hemiergis. Cartilaginous elements are indicated in black, precartilaginous in gray. A: In the forelimb, all three morphs share a common configuration with a phalangeal formula of X (far left) before developmental trajectories diverge. Top row: In the four-digit H. peronii, phalanges are added proximally to distally across all four digits until the adult formula of X is reached. Center row: In the threedigit H. peronii, phalanges are only added across digits II IV; only a metacarpal remains in the digit V position. Bottom row: In the two-digit H. quadrilineata, phalanges are added only to the central digits, although four metacarpals are present. The adult phalangeal formula of H. quadrilineata (2/2) is X B: Hind limb condensation sequences follow a similar pattern and adult phalangeal formulae are identical to those of the forelimbs for each morph. This study is the first to thoroughly document limb chondrogenesis sequences in any scincid lizard and the first comparison of limb ontogenies in a graded morphocline of any tetrapod. Previously, chondrogenesis sequences for only a few five-digit tetrapods had been reported (Sewertzoff, 1904, 1931; Steiner, 1922; Mathur and Goel, 1976) and not all of these ontogenetic sequences are complete. An advantage of studying limb chondrogenesis and reduction in Hemiergis is that closely related organisms in this case, four morphs of three species within a single genus are the focus. Moreover, one of these morphs has five digits, representing the putative primitive condition for the group (although phalangeal counts in H. initialis, 5/5, differ slightly from the primitive squamate configuration). Hence, studies of limb development in Hemiergis could provide insights into both general and derived patterns of lizard limb skeletal ontogeny. The limb chondrogenesis sequence for Hemiergis initialis (5/5) resembles those described for other pentadactyl lizards. Importantly, truncations of this program at intermediate developmental stages would yield incomplete digits, not fewer complete ones. Hence, the adult limbs of Hemiergis with fewer than five digits do not result from simple heterochronic truncations of a five-digit (or any other) skeletal developmental program. Ossification sequences in Hemiergis resemble those of other lizards as well and all cartilaginous elements formed are eventually replaced by bone, thereby eliminating ossification failures as a source of limb reduction. This investigation also identifies positional homologies between digits of different Hemiergis morphs. Morphological cues in adult Hemiergis, such as phalangeal formulae, can be misleading when assigning digit positional homologies. As discussed below, this developmental study confirms that digit I is indeed lost in H. quadrilineata (2/2) and H. peronii (3/3 and

13 LIMB DEVELOPMENT AND REDUCTION IN HEMIERGIS 223 Fig. 12. Carpal and tarsal ossification in Hemiergis. A,B: Independent ossification of distal carpals 4 and 5 in Hemiergis quadrilineata (2/2) juveniles. A: Dorsal view of wrist of HQ-001 (SVL 22.5 mm) cleared and stained for cartilage (blue) and bone (red). The earliest ossification center of distal carpal 4 5 (the cartilaginous fusion of distal carpals 4 and 5) is located directly proximal to metacarpal 4 and does not include cartilaginous distal carpal 5. B: Dorsal view of wrist of HQ-101 (SVL 30 mm). In this larger specimen, ossification of distal carpal 5 begins adjacent to but distinct from distal carpal 4. C,D: Tarsal ossification in Hemiergis juveniles. C: Dorsal view of ankle of H. initialis (5/5; HI-105) showing early ossification of the astragalus (arrowhead) within the tarsale proximale, the cartilaginous fusion of the astragalus and calcaneum. D: Ventral view of ankle of H. quadrilineata (HQ-101) showing secondary ossification centers in the astragalus (black arrowhead) and distal tarsal 4 (white arrowhead) within the tarsale proximale. Additional secondary centers of ossification are visible in the proximal metacarpals and distal tibia and fibula. as, astragalus; c, centrale; ca, calcaneum; dc, distal carpal; dt, distal tarsal; Fe, femur; Fi, fibula; H, humerus; mc metacarpal; mt, metatarsal; pis, pisiform; R, radius; re, radiale; T, tibia; tp, tarsale proximale; U, ulna. Scale bars 0.1 mm. 4/4) and that at least some portion of digits II V is retained. Interestingly, phalanges appear in digit V of several H. peronii (3/3) embryos (Table 1), representing an atavism or low-frequency polymorphism. The functional significance of different digit configurations in Hemiergis is difficult to assess without controlled studies of locomotion. However, a regres- sion analysis of limb and body lengths reveals that digit number is not linearly correlated with limb length. When coupled with behavioral observations of wild and captive Hemiergis and other reducedlimbed skinks (discussed below), these results permit limited generalizations about the functional correlation between digit number and limb length.